McCarthy T L, Ji C, Shu H, Casinghino S, Crothers K, Rotwein P, Centrella M
Yale University School of Medicine, Section of Plastic Surgery, New Haven, Connecticut 06520-8041, USA.
J Biol Chem. 1997 Jul 18;272(29):18132-9. doi: 10.1074/jbc.272.29.18132.
Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind IGF-I cAMP response element or any adjacent sequences. These results provide new molecular evidence that estrogen may temper the biological effects of hormones acting through cAMP to regulate skeletal IGF-I expression and activity.
胰岛素样生长因子-I（IGF-I）是骨重塑的关键因子。在成骨细胞中，甲状旁腺激素和前列腺素E2（PGE2）通过cAMP激活的蛋白激酶增强IGF-I的合成。在大鼠中，卵巢切除术后雌激素缺乏导致血清IGF-I升高和骨重塑增加，而雌激素治疗可逆转这两种情况。为了在分子水平上研究雌激素对IGF-I表达的依赖性调节，将原代胎鼠成骨细胞与雌激素受体（hER，以确保活性ER表达）以及由大鼠IGF-I基因启动子1（IGF-I P1）控制的荧光素酶报告质粒共转染，该启动子仅在这些细胞中使用。如报道的那样，1 microM PGE2使IGF-I P1活性增加了5倍。单独的17β-雌二醇没有作用，但剂量依赖性地抑制PGE2的刺激作用达90%（半数有效剂量约为0.1 nM）。这种情况在3小时内发生，持续至少16小时，需要ER，并且似乎具有特异性，因为17α-雌二醇的效力低100 - 300倍。相比之下，17β-雌二醇刺激雌激素反应元件（ERE）依赖性报告基因表达高达10倍。17β-雌二醇还抑制了仅保留cAMP依赖性基因激活所需最小启动子序列的IGF-I P1构建体，但不影响PGE2诱导的cAMP增加60倍。大鼠IGF-I P1中没有一致的ERE，这表明在通常增强骨骼细胞中IGF-I表达的cAMP途径中存在新的下游相互作用。为了探究这一点，使用IGF-I P1中的非典型cAMP反应元件通过电泳迁移率变动分析检测表达hER的成骨细胞的核提取物。单独的雌激素不会导致DNA - 蛋白质结合，而PGE2诱导出特征性的凝胶迁移复合物。两种激素共同处理导致凝胶迁移强度大大降低，这与它们对IGF-I启动子活性的联合作用一致。然而，hER不与IGF-I cAMP反应元件或任何相邻序列结合。这些结果提供了新的分子证据，表明雌激素可能调节通过cAMP起作用的激素的生物学效应，以调节骨骼IGF-I的表达和活性。
